Recruitment in randomized clinical trials: The MeMeMe experience

Introduction Recruitment is essential for the success of clinical trials. We are conducting a randomized clinical trial to test the effect of a Mediterranean dietary intervention with or without 1700 mg/day of metformin for the prevention of age-related chronic diseases, the MeMeMe trial (Trial registration number: EudraCT number: 2012-005427-32 ClinicalTrials.gov ID: NCT02960711). MeMeMe recruiting experience, highlighting strengths, limitations encountered and results is reported. Patients and methods Statistical analysis focused on the reasons for withdrawal according to the recruitment method (“active” versus “passive” criterion) and the time of withdrawal. Logistic regression models were used to explore the associations between the risk of withdrawal and sex, recruitment method, randomization arm, and with markers of compliance to the intervention, such as one-year change in body weight. Results Out of 2035 volunteers, 660 (32.4%) were recruited “actively” and 1375 (67.6%) “passively”. Among people who dropped out of the trial after randomization, there were 19.5% for the “active” and 22.0% for the “passive” method (p = 0.28). The risk of withdrawal was significantly higher in women (OR:1.91; 95% CI:1.17–3.12; p = 0.01), in volunteers older at recruitment (OR:1.25; 95% CI:1.07–1.45; p = 0.004), and in those with a higher BMI at baseline (OR:1.23; 95% CI:1.07–1.43; p = 0.004). Volunteers who lost at least 2 kg (the median weight change) in the first year of intervention were significantly less (53%) likely to withdraw from the trial (OR:0.48; 95% CI:0.30–0.75; p = 0.001). Conclusion Our findings suggest that the “passive” recruitment method was more effective than the “active” one to advance recruitment. The benefits of “passive” recruitment hardly outweighed the drawbacks. Trial registration Trial registration number: EudraCT number: 2012-005427-32. ClinicalTrials.gov ID: NCT02960711.

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The special clauses set out in Article 7 shall take precedence over any other provisions of ±is grani agreemeni.   Italian, bom in 1944, MD (1969) PhD in pathology (1975, epidemiologist, has been active on cancer research and chronic discese prevention for almost 40 years, witti aver 350 peer-reviewed scientific papers. In the 1970s FS established the first Itatian cancer registry in the Varese Province (IARC Cancer Incidence in Five Continents, Vokmes V io /X) on the basis of which ho carried out severa[ population basati etiological studies on tobacco, alcohol, occupation and envirorimental pollution. As soon as the registry started producing survival data ha promoted the EUROCARE project, a conoerted action between virtually al] European cancer registries aimed al describing survival differences, understand the reasons why they exist, and help preventing inequalities (Berrino et ai Survivai of Cancer Patients in Europe, Ì.4RC Scientffic Pubiications 132, 1995, and151, 1999;Annais af Oncoiogy (suppiement 5), 2003 (Gatta stai Cancer 200089:893, and 2002, 95:1757Sant et al, Cancer 2004, 100715, Ciccolallo et al. Gut 2005Cofeman sta! 2008 Lancet Onco( 9:730) and showed that the differences are Iargely due io earlier diagnosis in the US, and nel te stage specific more effective treatment.
The ORDET experience was pivotal lo the development of the EPIC (European Prospective Investigation into Cancer and nutrition) multinational cohort, the Iargest prospective study with biologica] repository ever undertaken. FS co-authored severa[ EPIC papers on diet and head and neck, stomach, colon, lung, breast endometrium, ovary, prostate, and biadder cancer in relation to diet, anthropometrio and metaboljc and hormonal factors. Given this long lasting experience, FS co-ordinated the section on breast, ovary and cervix cancer for the American Institute far Cancer Research / World Cancer Research Fund systernatic literature review (A1CR4VCRF Food, Nutritian, Physicai Activity and the Preventian or Cancer: a Globai Perspective, 2007) In the 1990s FS realized that the accumulated evidence on hormones, lifestyle and breast cancer was sufflciently strong io juslify the undertaking of randomized dietary intervention studies on women ai high risk because of high serum androgen Ievels or other endocrine-metaboiic high risk traits. The rationale was that western sedentary and hyper-caloric diet Iifestyle is associated with insulin resistance, and insutin stimulates The synthesis of androgens in the avary and inhibits the liver synthesis of St-IBG and IGFBPI and 2, thus increasing The bioavailability of both sex hormones and IGF-l. Tbc DIANA (Diet and ANdrogens) trials proved that a comprehensive dietary modification aiming at iowering insulin Ievels, based on Mediterranean traditions and macrobiotic principies (Iow fat, reflned carbohydrates and animai products, high who!e grain cereals, legumes and vegetabies) decreases the bioavailability of sex hormones and severa[ growth factors (Serrino et al 2001Cancer Epidemia! Biomarkers Prev 1025, and 2006 Ann NY Acad Sci 1089:II0) and suggested that can decrease breast cancer recurrences (Berrino et al. 2005 inti Cancer 113:499). in the latter study high serum testosterone ievels, high 6Ml, the presence of metabolio syndrome and the contemporary presence of high IGFA and PDGF were signitoant negative prognostc factors (Pasanisi et al, 2005mt i Cancer 119:236, and 2008Cancer Epidemie] Biomarkers Prev, 17:1719. Actuaily The sane hormonal and metaboIic indicators of breast cancer risk eisa appeared io effect breast cancer prognosis. The success of the DIANA studies, which were based on a few hundred patients, prompted the planning of a large scale randomized trial of adjuvant diet and physical activity to reduce recurrences in 1510512013 breast cancer patients with endoorine/metabolio pattern suggesting high 4Y4 recurrence risk (either high testosterone, or insulin, or presence of rnetabolic syndrome). The trial has recruited 2000 patients and the foilow-up is ongoing ( Viliarini, stai, Lifesty!e and breast cancer recurrences: The Dt4NA-5 trial. Tumori, 98: 14-31, 2012).
FB has no interests of any kind that could reasonably be perceived te affect his scientific objectivity. His scientific acflvity is motivated solely by his passion far cancer prevention.

Funding ID
There Is and there wili be no funding overiap with the ERO grant requested and any othar source offunding fo, the seme activities and costa that aro foreseen in this project.
In relation te this propose[ FB intends io apply for a further grant io study epigenetio profiies before and after calorie restriotion and nietformin treatment. Such a study would invoive anly a few hundred persons valunteering for the present MeMeMe project, and they will be requested te sign an independent informed conseni. A previous applicatian te the Italian CARIPLO Foundation faiIed; the foliowing bax reports its executive summary: treatment The western lite style, characterized by a hyper-calorio dici rich in fat, refined carbohydrates and animai proteins is assaciated with a high prevalence of obesity, diabetes,metabolic syndrome (MetS), insukn resjstance, and elevated haematic levels of growth factors and, in women, sexual hormones. These factors aro also associated with a high risk for some of the mesi frequent tumours in western population, such as coiorectal and breast cancers (CRC and BG), and te a higher reiapse risk in patients that have already been diagnosed and treated for these diseases. Dozens of animai experiments have proven beyond reasonable doubt that moderate calorie restriction without malnutrition (CR) is the most potent dietary intervention for improving insulin sensitivity and other markers of MetS, preventing Gancer, and pralonging lite. A sustainable CR can be obtained with traditional Mediterranean diet (MedDiet), characterized by highly satiating staple food, such as whole grain cereals and pasta, legumes, seasonal vegetables, nuts, olive oil, fruit, moderate dine, and occasionally animai products, mainly flsh. MedDiet prevents MetS and is associated with lower incidence of several cancers, ineluding BC and CRC. Diabetic patients have increased risk of cancer, but severa] observational studies showed a signilicantly lawer risk among thase treated with metformin (MET), a CR mimetic.
There is increasing preclinical evidence suggesting that CR and MET favourably madify the risk ol cancer through epigenetio mechanisms. We intend lo explore such mechanisms in humans. We will profit of our largo prospective cohort studes with biological repository (plasma, serum, butfy coat), in which we ere failowing up tens of tliousands of healthy women for the occurrence of canoer, of our clinical cohorts of [housands of BC patients, and of our randornized controlled studies of dietary intervention and chemoprevention, in whioh we ere treating volunteers, either healthy but with MetS (for the prevention ol cancer), or with previous diagnosis of BC (far the prevention of metastasis), with either CR or MET.
We will analyse epigenetio traits (DNA methylation, histone modification, and miRNA expression) before and after treatment with CR, MET, or both, in women with or without MetS, abdominal obesity, and different lifestyle and dietary habits. We will then evaluate if the epigenetic changes discovered in women enrolled in our dietary and chemopreventive trials ere associated with the subsequent incidence of BC and CRC in our epidemiological cohorts, and with the occurrence of metastases in our clinica[ cohorts (taking into account in the study design and in the analysis the classica[ epidemiological and clinico-pathological risk factors). We will examine in particular the incidence of metastasis in women with triple-negative BG. In a sample of wòmen we will study the epigenetic traits in epithelial breast celle obtained through nipple As it recently ernerged that MET can direotly suppress the proliferation of tumour initiating celle (TICe or cancer stem celi), we will also test this hypothesis in vivo treating witil MET imrnunodelloient mice xenotransplanted with human CRC cells enriched in TICs, and we will analyse the induced epigenetic chanqes, which will be than compared with that observed in women subjected to the different treatments.
The new study we intend te apply for is not a hunlan gene-mapping study; we aro not interested in identifying any allele that predispose to an illness, nar in carrying out family studies; we have no commercia[ interest nor partners. Il is just a study on the epigenetic effects of calorie restriotion and of Ireatment with metformin te be carried out on a voluntary sample of participants in the main study. Depending on the discoveries of such epigenetio research, however, the analysis of a few speciflc genes, e.g. genes whose variants may affect the effectiveness of calorie restriction or mettormin, such as AMPK and LKBI, may become relevant te the interpretation of the results. We wilI describe in terme understandable te the participants the nature of this new epigenetic/genetic study, in order te give them an opportunity te decide whether or not te participate. We wilI guarantee that ali studies involving their samples will tre dono anonymously, and That no one oiitside the 4 1510512013 study team will have accesa to the individuai data. We will respect participants decision to consent or not consent the use of their samples beyond the primary objective of exploring the epigenetio ohanges. In case they acoept we will be available to inform theni on the signWicance of the genetio lraits that rnay emerge.

Background and objecti e k414
Worldwide, we aro entering a period af unprecedented global aging, and age-related chronio noncommunicable diseases (ArCD) and related disabilities aro projected te intiate into an unprecedented econonlic and acciai challenge. AJCD aro Iargely due te modifiable western litestyle factors. Previous Iifestyle and chemopreventive trials te prevent ArCD have generaily been unsuccessful. Most were based on the supplementation or the avoidance of a singie or a few nutrients, and most failed or ended with dubious results. The ATBC, CARET and SELECT trials, which supplemented antioxidant vitamins or minerais io prevent cancer, were interrupted because of increased cancer incidence or serious side effects. Also in the cardiovascular tield preventive trials supplementing antioxidant and other vitamins showed little or no cardiovascular benelit. Nevertheless, the present epidemiological and biological knowledge of ArCO makes a new generation of preventive tria!s promising, feasiblo, and worthwhile. Progress in ArCD biology has been extremely fSt in the last few decades, pointing out te the relovanco of complexity as opposed lo the previously dominant reductionist view of nature. Emerging data demonstrate that the effect of lifestyle factors Is largely mediated by epigenetic changes, with a cross-talk that is largely te be unravelled between DNA methylation, histone modiflcations, and noncoding RNA. kCD share severa] risk factorsovereating, sedentary lifestyle, industrialiy processed food, excess body weight, insulin resistance, chronic inflamrnatory status, and a predisposing (e.g."thrifty') genotweand we hypothesize that a many faceted intervention is needed to favourably effect the compiex biological system involved in tileir development. A single agent or factor acting on a singie ora few pathways might actually fafl because of the existence of redundant vicarious pathways, or even be dangerous because of its interference with potentially preventive pathways.
The aim of the present study l a te evaluate the effect of a comprehensive life-style intervention (including moderate physical activity ami Mediterraneanlmacrobiotic diet with moderate calorie and protein restriction), and of troatment with Metformin (a calorie restriction mimetic drug) for the prevention of ArCD. The plan is te carry cui a randomized controlled trial on men and women, 55-74 years of ago, at high risk of ArCD because of metabolic syndrome.
Metabotic syndrome (MetS) The malor metabolio risk factor for the development of ArCD is the MetS, whose prevalence in adult population ranges between 20 and 40% in different western countries. MetS is defined as a clustering of cardiovascular discese (CVD) risk factors of metabolio origin. The World Flealth Organization l-IO) was the first te propose criteria far diagnosis of the syndrome, followed by severa] other organizations. The mesi frequently used criteria aro those of the National Cholesterol Education Program Adult Treatment Fanel III (2001( Jama.265:2486, which defines MetS as the presence of three or more of the foliowing conditions: Waist circumference equa] te or greaterthan (k) 88 centimeters in women or 102cm in men, Glucose 5.6 (mmol/l), Triglycerides 1.7 mmol/l, HDL-Choiesterol <1.0 (men) or <1.3 mmol/I (women), blood pressure k 130185 mmHg. Several other thresholds were subsequently proposed by other agencies. The international Diabetes Federation, far instance, recommended that the threshold of waist circumference te detine abdominal obesity in people of European origin should be 80cm for women and ~ 94 cm for men. Te siniplify the instruction for the self-selection of participants our threshold for waist circumferenoe vili be 85 cm for women and 100 cm for men. As suggested in a meeting of severa] major organizations in an attempt te unify criteria we will consider as MetS components also the presence of speciflc treatments far lipid metabolism and for previously diagnosed hypertension, and the cut point for elevated fasting glucose will be 2100 mgJlOOmL (Harmonizing the Matabolio Syndrorne 2009 Cireulation 120:1640).
Classical studies showed that the MetS is accompanied by a two-fold increase in the risk of CVD and a uvefold increase in the risk oftype 2 diabetes mellitus. More recently several studies showed that MetS is also associated with increased risk of cancer, neurodegenerative diseases, non-alooholic fatty liver discese and cirrhosis, chronic kidney discese, galibladder stones, kidney stones, osteoarthritis, gout, prostatic hyperplasia, thromboemboiic disease, psoriasis and other chronic conditions. MetS related canoers include colorectal, liver, gailbiadder, pancreas, breast, cervix, endometrium, ovary, kidney, esophagus (adenocarcinomas), and probably prestate and lung cancer, representing two thirds oftotal cancer incidence. The mechanism is hkelyto involve hyperglycemia, hyperinsulinemia, and its effect on the synthesis and bioavailability of sex hormones, growth factors, and prointlammatory oytokines, lncreased leptin and decreased adiponectin levels also disrupt homeostatic signaling pathways involved in celi proliferation, survivai, cell-cycle regulation, and angiogenesis.
Calorie restriction (CR) Since the beginning of the Iast century, moderate CR without malnutrition (an experimental mode in which test animals receive a Iower-calorie diet than ad Iibitum-fed controls) has emerged as the most potent dietary intervention for prolonging lite and preventing ArCO, including cancer, diabetes, artheriosclerosis, cardiomyopathies, autoimmune diseases neurodegenerative diseases, respiratory and kidney diseases. Results in rhesus monkeys also showed that Sarcopenia, a serious health concern associateci with advancing aging, was partially prevented in the CR group, as well as the bss of Thell fijnction and gray 8 1510512013 matter volume. Humans undergo severa] of the same adaptations to CR'Itcq-laboratory animais, inoluding lower Ievels of cardiovascular risk factors, inflammatory oytokines, thyThtdffmones, insulin, leptin, sex hormones (in women), and higher [svela of steroid hormone-binding proteins, adiponedin and other hormones that suppress inflammation, better left ventricuiar diastolic function, and lower carotid intimamedia thickness than healthy ago and sex matched controls. Recently, CR and a pharmaoological CR mimetic (rapamycin, a macrolide ttiat produces a gene expression pattern that overlap Uiat seen with CR) were shown to extend lifespan and delay canoer incidence in mice that were already in lato middle age when treatment began (up to 19 months, suggesting that starting Iifestyle interventions on men and women having broadly the sanie ago -above 55 years orso -could be promising).
The responses Io CR involve numerous pathways and intricately linked mechanisrns that are far from being completely understood. Among the niechanisms by which CR may reduce metabolie and neoplastic risk tbc well established ones aro: A) the decrease of the ATP/ADP ratio, with the subsequent activation of the adenosine 5'monophosphate-activated protein kinase (AMPK), a sensor of energy availability (the fuel gauge"), whose activation iflhibits energy consuming processes, including celi proliferation, and activates various other potentially preventive pathways.
Various posttranscriptional aiterations relevant to CR are being discovered, e.9, miR-217, which nhibits SIRTI, and promoter methylation profile of leptin and TNF-alpha during weight bss.
Several studies suggested that most of the effect ci CR is due to protein restriction, wliich seems necessary, at ieast in humans, to reduce the plasma concentration of IGF-1, whose high level is associated with increased risk of subsequent cancer. In western countries people eat about the double of the RDA of proteine (15-16% of calories vs. 83%) and reducing protein intake may be more sustainable than reducing total calorie intake. Both objectives, however, can be obtained witli traditional Mediterranean diet and with macrobiotic diet.

Mediterranean diet (MedDiet)
Classica] studies showed that MedDiet (le. a diet that was largely based on unrefined cereal products (mainly bread and pasta), pulses, vegetables, olive olI, nuts, fruft, moderate wine, occasionaily fish and cheese, and rarely other animai products) is associated with lower risk of CVD and diabetes. Subsequentiy it became clear that subjects with the highest adherence to a fraditionai MedDiet have lower cumulative incidence of MetS, and of several ArCD than those with the iowest adherence. Randomized ntervention triabs showed that MetS can be reversed with MedDiet, witli up to 69% rechiction of MetS prevalence afier 2 years of diet. Severa[ other trials supported favourable effects of MedDiet on body weight, abdominal adiposity, plasma hpid protile, and endothelial fijnction. Lower adherence to MedDiet Is also associateci with the development of cognitive impairment and of Parkinson and Alzheimer disease.
Several observational studies on dietary patterns showed that Medfliet Is signiflcantly associated with lower risk of breast cancer, colon cancer, melanoma, coronary hearth discese), stroke, diabetes, Alzheimer disease, and overall mortality. -Whoie grain cercaI intake and macrobiotic diet Several studies showed consistent!y that persons who ingest three or more portions of food per day based on wholegrain cereals and whole-wheat bread have a 20-30% lower risk of cardiovascuiar diseases and type-2 diabetes Than subjects who ingest Iow quantities of cereals. Two recent largo cohort studies on the intake dietary fibres and mortality, the EPIC study in Europe and the NCI-ARP study in tbc IJS, consistentiy showed that an high intake of cereal fibres Is associated with lower mortality freni cancer, cardiovascular, pulmonary, gastrointestinal, and infectious diseases. The isolated administration of whole grain cereal componente (flbres, vitamins, minerale), or the sane arnount ai fibre from retlned grains, do not seem te elicit any protective effect, suggesting additive or more than additive influence of cereal constituents on health, the so cailed 'food synergy" Whole grato cereals (mainly brown rice, millet, barley, oats) and ocdasionafly buckv4eat and v4ieat pasta, are the basic component of macrobiotio diet, which inciudes also 20-30% iocaily grown vegetables, 5-10% beans, inoluding traditional soy products, sea vegetables and occasionally fruits, nuts, flsh. The 2007 WCRF/AICR recommendations for the prevention of cancer (wvv.dietandcancerreport.org ) broadly coincide with the macrobiotic recommendations of avoiding the habFtual intake of extremely "yin" food, such as sugared and alcohoiic beverages, as weli as of extremely "yang" food, such as processed meat, saity food and red meat, wliile the centrai recomrnendation is te "Eat mostly food of piant origin, with a variety of non starchy vegetables and of fruit every day and with unprocessed cereais and/or puises within every meai", vdiich is also the basio charaoteristic of the MedDiet, as weB as the stapie food ofniostpopoiations before the industria] revolution: cuscus wfth chickpeas io north 1510512013 Africa, rice with soy products in Asia malze with black beans in centra eri illet with peanuts in black Africa.

Physical activity
In observational studies, moderate physical activity is associated with lower incidence of CVD, cancer, lower prevalence of MetS parameters and iniproved diabetes control. Several randomised studies of lifestyle education that included both dietary modificalion (mainly calorie restricted, low fat, and high fibre diets) and physical exercise consistently showed a 50% lower incidence of diabetes in persona with irnpaired glucose tolerance or impaired fasting glucose.

Mettormin (MET)
MET, an orally administered biguanide drug widely used io treat diabetee, has been shown to prevent diabetes in people with glucose intolerance, to reduce cardiac risk factors and prevent CVD in diabetics, and to improve clinica] outcomes in patients with heart failure. lmproved glycemic contro] in patients treated with metformin Is associated with a decreased risk of diabetes related cardiovascular end points and ali cause mortality when compared to conventional therapies that Iower blood glucose to similar levels. Therefore, the cardioprotective effects of rnetformin cannot be attributed Io its anti-hyperglycemic effects alone, but may be also related to the actions of mettormin Cn Iipid nietabolisni, vascular smooth muscle proliferation, cardiomyocyte calcium handling, endothelial funotion, hypercoagulation , ami platlet reactivity. Such pleiotropic effects of metformin ere largely mediateci by the activation ofAMPK, a protein that is normaily activated in response to CR, vIiich stimulates fatty acid oxidation, promotee glucose transport and glycolysis, inhibits neogiucogenesis ami bolh triglycerides and protein synthesis.
Diabetes is associated with increased cancer incidence. F-!owever, several observational studies (mainly retrospeotive cohort studies) showed that diabetio patients treated with metformin bave a significantly bower risk of developing cancer (ali sites combined) than those untreated or Ireated with other drugs. MET may reduce cancer risk Through two main mechanisms: C) reducing insulin resistance and therefore insulin, testosterone and IGF-I concentration in blood, and 2') activating AMPK, thus mimicking the effect of CR. AMPK activation results in downregulation of mTORCI and IGFI/Akt pathways, and in p53-mediated celi cycle arrest. MET also inhibits IRS-1, thus preventing the feed-back activation of Akt that may follow mTOR inhibition.
MET is a very safe drug used since 50 years to treat diabetes ami more recently to prevent diabetes in people with glucose intoberance. As phenformin, a drug acting through the sanie mechanism, caused several cases of bactic acidosis, the issue was raised that also MET might cause such a serious side effect. ps stated in a recent Cochrane systematic review, however, There is no evidence from prospective comparative triais or froni observational cohort studies that mettormin is associated with an increased risk of bactic acidosis, or with increased bevebs of lactate" (Cochrane Database Syst Rev. 2006 Jan 25; (/):C0002967). Neverthebess we will carefubly exclude from the study any person with chronio hypoxemic conditions that may be associated with bactic acidosie, such as cardiovascular, renal, hepatio and pulmonary disease, or under treatment with drugs that might interfero with MET clearance (see operative protocol). A physician will be avaibable 241b124 to answer questions on the phone if the participants do noi feel well for wtiatever reason

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Molecular patliways that mediate the preventive effetftR and of MET Specifie DNA methy!ations and histone modificationa, usually associated with pro-inflammatory micro-enviranment bave -been implicated in several PJCD (cancer, CVD, obesity, COPD). Global hypomeThylation and site-specific hypermethylation are common featuree of human tumours and have been detected also in atherosclerotic Iesions. Diet can profoundly alter epigenetic patterns, but the causai link between diet and epigenetics in the development of human disease is stili poorly understood. A challenging researoh jjeld is developing te determine which adverse epigenomic marks aro reversible by speciflc drugs, nutrients, or Iifestyle changes. Besjdes CR, aver 50 bioactive phytochemicals have been demonstrated to be active on DNA methyltransferase, or histone acetylase/deacetilase. Experimentai studies on animale or cultured human celi linee support Their role in the prevention of AjCD, but have often been conducted ai concenfrations far beyond those documented in humans. It is very difflcult to predict from these resuits the effects of these substances on disease prevention in humane. There Is inoreasing evidence, however, that continuous exposure ai physiological concentrations can remodei the epigenome in a cumulative fashions. We will create a biorepository of biood samples, collected ai tvio time points (before randoniization and after one year), te can -y out mechanistic studies on the epigenetic changes induced by iifestyle modilication and MET treatment. With independent financial support we intend io systematicaliy explore the epigenetic changes induced by CR, MET and CR * MET. A financial request has been submitted to the Italian CARIPLO Foundation (see Funding ID).
Importance ot the project Our jmportant, innovative, and bold hypothesis is that we con prevent, or ai Ieast postpone, the occurrence of virtually ali ArCD through a comprehensive change of several aspects of western litestyle, supported by a chemopreventive agent That acts on the same genetic pathways affected by Iifestyle factors, and that we will obtain a very high compliance. Our confidence derives Thom The metabolio success of previous dietary intervention studies on a few hundred subjects (the DIANA trials) and Thom the preliminary results of our ongoing DIANA-5 filai, in which we succeeded to recruit 2000 breast cancer survivors ttiat have been randomized in a Ufestyle intervention and a contro[ group to study The effect of moderate CR and increased physical activity on the incidence of recurrences Changing so many aspects of Iifestyle, and examininu so many outcomes, we will not be able to understand what has caused what. This may be irrelevant from a pragmatic public heaith point of view, but is certainly relevant Thom an explicative point of view. Beside the basic Intention te ireat" analysis of the trial, therefore, we wili analyse the outcomes aiso as a function of compliance with The different litestyle recommendations, and oftheir association with MET treatment. With repeated 241h food diaries, far instance, we wili be able te disentangie the mie of pratein restriotion fran that of CR, and to expiore the effect of specitic foods. Results will noi have The strengffi ai a randomized design, but wili help mechanistio interpretation. Continuing the observation beyond the presently pianned 5 years we wiil aiso have the power to study severai speciflc diseases. b.

• Methodology
Main study hypothesis Our hypottiesis Is that a substantial fraction ofArCD incidence and mortaiity, ai the order of 25-33%, oould be prevented through a) a sustainable comprehensive change in iifestyle, inciuding moderate calorie and protein restriction, Mediterranean/macrobiatic diet, and physical activity, and b) chemopreventive treatment with metformin, a drug whose mechanism include the activation of the same genetic pathways that are activated by CR and physicai activity. An anciiiary study wiil examine the epigenetic changes induced by the above treatments.
Study design Phase ID randomized controiled trial on men and women with MetS to test the hypomesis that comprehensjve iife-style changes andlor metformin treatment prevent ArCD.
Design: 2x2 factoriai: 2,000 voiunteers wili be randomized in four equa] groups of 500 each, and aliooated te the foliowing treatments: METEORMIN  Main outcome: Totai incidence af kCD, including cancer, coronary heart disease, stroke, diabetes. Secondary outcomes: changing prevalence of MS and !te metaboiic and anthropometric componente, total mortaijty, tatal cancer incidence, incidence of diabetos, cardiovascuiar diseases, myocardiai infarction, stroke. These outcomes have been seiected as major objectives because they aro fairly easiiy identillabie on clinicai charts according te standardized criteria. Qther MetS associated ArCD, however, vÀil be registered in Il .,Y \ MeMeMe 322752 1510512013 the f&Iow-up and may eventually contributo to the overali discese burden '%'uated. Recruitment: Potentially eligible persone wili ho recruited through cancer screening programmes, generai practitioners, clinica[ units dealing with overweight ami rnetabolic diseases, and the media. Interested persons will be invitati to attend conferences in which we will illustrate in detail the study design ami its implications. Paper tape n -'easures will be availabie at the conference to help attendees to check their waist circumference, which is the basic eligibility criteria. Those above the threshold and without any obvious reasons not to be included, wili be asked to sign The informed consent form, and will be given an appointment far the iirst clinic visit and blood sample donation. To avoid drapouts far gastrointestinal discomfou -t we will treat ali volunteers with haif the planned dose [or ane month in order to exclude intolerant subjects before randomization. Parlicipante randomized in the intervention group will continue to take haif a dose for one month and then shift to the fili dose. Baseline requiremonts Participants will be requested: -to sign the informed consent to participate in the study ami to follow the presGribed treatment ami Iifestyle changes, to provide information on their health status and intervening diseases, and to allow the study officiais to contact their treating physicians, to consult clinical notes and to examine biood samples far metabolic, genetio and epienetic studies, even after the end of the active intervention period to fili in a questionnaire on medica[ history and risk factors (the EP1C-Italy questionnaire) -to undergo an anthropometric visit (height, weight, waist and hip circumference, blood pressure, body fat and lean mass measured with Tanita impedance device, atbaseline and after I year) -to donate a 20 mi blood sample (at baseline and after I year) -to fili in 24h dietary and exercise recali forma (either directly or on tbc phone) every 2 months in randomly chosen days. Participants will be informed that they con retire from the study at any moment, but recommended natta do so because il would damage the interpretation of results.

Randomization
As soon as ali the baseline data ere entered, ami the eligible partioipant confirms his/her willingness to go on, a computer system randomly estab!ishes the study groups, balanced far gender ami ago « 65 years vs. >65 years), and gives instruction to the pharmacy to preparo the drug bottles. The assignment is immediateiy recorded in tbc database ami not subject to alteration. Prescriptions: A) 2 tabiets per day, one at breakfast (or Iunch) ami one at dinner, of either metformin (two 850 mg tabletslday) or placebo (twa identical tablets) according to tbc blind assignment. In case of gastrointestinai discomfort occurring after randomization participants wiii be asked to continue witii a single tablet. In our pilot trial such side effect occurred in 10 cui of 80 participants: 7 ofthem stopped taking the pilI and 3 continued ai half the dose. Subjects stopping taking the pilis wili be kept in the study, but we wili increase the study size by 10% to partly compensate the power bss. B) participation in the Iife-styie intervention activities.
Ali participants will receive generai lifestyie recommendations for tbc prevention of cancer and cardiovascuiar diseases, inoluding smoking cessation counsefling, and those randomized in the hfe-style intervention group wili be offered to participate to four kitchen courses and wili then be invited monthly for iunch or dinner, physicai activity sessions, ami nutritionai counseihng to strengthen their commitment over The whole duration of the study. Suoh a scheduie proved to be sufficient to obtain signiflcant reduction of body weight (3 1Kg on average atter one year) and of MetS parameters in our ongoing randomized trial ofiifestyle modification in breast cancer patients.

Organization
We pianto caro] 25-30 volunteers per week over twa years (25 perions per 40 weeks per 2 years = 2,000). Fasting bload samples will be collected between 8 an 9 mm. Glucose, total and HDL cholesterol, and triglycerides will be immediately measured. We will then prepara 10 aliquots of senini (4 aliquots), plasma (3), red blood celIs (I), buffy coat (2), tu be preserved al -80°C. The blood soliection protocol is compatible with future categorization of genetie and epigenetic variants (we have tested il for n11RNA and DNA methylation). For a 10% random sample of participants we will extract immediately tbc RNA far gene expression studies.
Intervention and control groups will be separately invited every 6 manths through SMS messages to refill the tabiet botile and for a short conference. In such occasion we wili count the lettover tablets and participants will fili-in a questionnaire on any health events that may have occurred in the interval.
We have deveIoped a very efflcient system far communicating to study participants through SMS. We v411 uso the Milan national Cancer Institute facilities far blood coilec.tion and sample storage, laboratories far biochenilcal, and molecular biology determinations, open air physical exercise, gymnasium, teaching rooms, and dedicated ktchen and restaurant with 60 places far cooking cIsses and common meals.

Lifestyie intervention
The intervention ama ai increasing physical activity, controlling weight, and promoting nutrient-rich (Le. 100% RIDA for al] nutrients) moderately calorie-restrioted and protein-restricted iow-glycemic index diet. As far physical activity, the goal of the intervention Is to schiavo ami maintain regular participation in a moderate intensity physical activity (approximately Sto 5 METs) program of 210 minutes over at cast 3 days /week (30 min OD average per day). During the first four months, one group physical activity teaching session per month will be oftered. Far self-monitoring ami conipliance enhancement, study participants will be given a stepcounter and asked to periodically fili-in 24h physical activity diarìes. Persona who wish to take up vigorous sports will be encouraged to do so after cardiorespiratory fitness testing. For tliose who do not progress to more vigarous activity, the focus will be on niaintaining moderate intensity activities, such as walking.
To contro] weight, participants will be encouraged to includo whole grains and high-fibre vegetables, which add bulk and volume, in every meal, lo choose cooked cereals rather than white bread or dry crackers, to eat vegetables or a soup before eatlng higher energy foods, to choose fresh fruit rather than juice, to eat muta at least every other day, and ta avoid potatoes, sugary drmnks, commerciai sweets, white bread and other produots based on refined flours, high protein mea!s, and energy dense industrial foods. In order to reduce glycaemic and mnsulinemic response, recommendations wili include: reducing calorie intake, through the preferred consumptions of highly satiating foods, such as unrefined cereals, legumes and vegetabies reducing high glycaeniic index food, such as reflned flours, potatoes, white rice, corn flakes, and high insuUnemic foods, such as sugar and mulk, preferring instead whole grain rice, bariey, millet, oat, spelt, quinoa and buckwheat, legumes (any type including say products), seasonal vegetables (any type, except potatoes) -reducing sources of animai saturated fat (red and processed meat, milk and dairy products) preferring instead unreflned vegetable fats, such as olive ai[, nuts and oleaginous seeds reducing pratein intake, mainly animai protemn (except lish), down to 10% oftotal calorie intake eat niostly food of plant origin, witti a Vide variety ofseasonal products.
We vili teach how to prepare traditional Mediterranean and macrobiolic gastronomic dishes (heaithy, satiating, palatable and easy to prepara) based ori whole grain cereals, durum wheat pasta, ami legumes, seasoned with vegetable sauces and httle fat; and competitive cakes and cookies without sugar, milk, buffer, and reflned flour, using instead dried fruit, such as raisin and apricots, oleaginous secca, soy milk, cereal fiakes and unrefined flour. We will uso dietary sources of anIi-intiammatory food, such as unrefcned cereals, smali size oily lIsh, seaweeds, turmeric, greon tea, olive oil, wild berries and other quercetin rich vegetables, such as capers, apples, onion, horseradish, broccoli and other greco vegetabies, as well as other vegetabies rich in phytochemicals that have boen shown in vitro to inhibit Akt (which is critical to both cancer and atherothrombotic diseases) and other relevant genetic pathways, such as, genistein, morin, apigenin, deulnidin, lutealin. The principle, however, Is to avoid any excess and to guarantee the intake of every potentialiy preventive bioactive phytochemicals including in the menus a wide variety of seasonai vegetables and fnjits. We have recently pubhshed a book on the dietary prevention of cancer (Villarini and Allegro, Prevenire I tumori mangiando cdii gusto, Sperlung & Kupfer 2009) which include the recipes that will be taught io the lifestyle untervention group. 1510512013 FolIow-up We will actively oontact the participants far the whole duratìb&kthstudy, and obtain copy of relevant clinical records. The availability of an efficient regional hospital discharge diagnosis information systems, ami af an accessible national death index will make follow-up feasible and complete. The Lombardy region, moreover, is almost completely covered by a network of canoer registrios, whioh will allow lo retrieve pathology reports. We will retrieve records for ali NCD but we will flrst concentrate the analysis on cancer, coronary heart disease, stroke, and diabetes, for which we have aiready used the above follow-up procedures in several previous studies. Far the registration of cancer cassa we will follow the rules and definitions af the European Network of Cancer Registries (available ai v,w.iarc.fr ). We will register cancer site, morphology, TNM stage, grade, and, for breast cancer hormonal receptor expression. Far the definition of diabetes we wiil follow the criteria of lire American Diabetes Association: a) Symptoms of diabetes plus casual plasma glucose concentration ~200 mgldl (11.1 mmol/l). Casual la defined as any time of day without regard to time since last meal. The classic syrnptoms of diabetes include polyuria, polydipsia, and unexplained weight [ osa, or b) FF0 I26 mgidl (7.0 mmol/l), oro) 2-h postload (75 g) glucose U00 mgldl (11.1 mmolII). d) In the absence af unequivocal hyperglycemia, These criteria should be confirmed by repeat testing on a different day. Suspected deaths from Coronary Heart Disease (CHD) and Cerebrovascular Disease (CBVD) will be identifled from mortality files when the oodes ICIDI 0 120-125, 150,170 in associatian with 120-125), or, respectively, the codes [160][161][162][163][164][165][166][167][168][169][110][111][112][113][114][115]146,149,and 170 in association with 160-169), aro reported as underlying cause of death. Fata[ CHE) and CBVD wilI be assigned only after verification against hospital discharge and chnical records. Persona with suspected CHD or CBVD will be identified on hospital discharge forma with ICD9-CM 41 0-414 codes or procedure codes for coronary revascularization, and, respectively with codes 342, 430-434, 436-438 or procedure cades for carotid revascularization. Clinical records will be retrieved te verify lire diagnoses. CHE) will be considered verified when acute myocardial infarction, acute coronary syndrome, or coronary revascularization ara present, backed up by information on onset symptoms, Ievels of cardiac enzymes and troponins, and electrocardiogram data coded according to The Minnesota Code. bschemic thrombatic stroke will be diagnosed when brain infarction is mentioned in the diagnosis and/or confirmed on the basis of imaging exanis (computed tomography or magnetie resonance imaging). Hemorrhagic stroke will ho diagnosed when cerebral hemorrhage is nientioned in the diagnosis or conlirmed by imaging. For other potentially relevant chronio diseases, such as neurodegenerative diseases, arthritis, gout, fatty liver disease and cirrhosis, galibladder and kidney stones, autoimmune diseases, which are not formai objectives of the application, we will examine the feasibility to collect reliable and standardized clinica[ diagnosis in the course of the study.

Blood examinations
Ai baseline and after one year we will examine the metabolic rnarkers of MetS.
-Further analysis of insulin, IGF-I, GF-BPs, PDGF, sex hormone, hsCRP, and infiammatory cytokines, will ho carried out in appropriata samples of participants from the third year onward. We will examine baseline ami 1year samples in the same batch. We will study the effect of one year CR, one year MET treatnient, or their combination, in subjects with high and Iow compliance (deflned by changes in body weight, waist circumference, and 24h recali diaries). Eventually, we will examine the change in these parameters in people who did or did noi develop speciflc ArCD with a case contro] design. Changes in epigenetic profiles will be examined in the frame of companion project far which we ere seeking independent financial support.
The study team has extensive experience in large-scale prospective cohort studies with biologica] repository (we ere following-up 50,000 persona in Italy), and in dietary intervention studies (we succeeded lo randomize 2,000 breast cancer survivors into a Iifestyle intervention and contro] group ami we managed to keep a very high dietary compliance of the intervention group.

Analysis
The main analyses will be by intention to treat. We will compare the incidence ofArCD in: 1,000 persona treated with MET with the 1,000 treated with placebo 1000 persona randomized in the Iifestyle intervention group with the 1,000 in the contro[ group 500 persons treated with bolli MET and Iifestyle intervention with the remaining 1,500 1,500 persona treated with MET or Iifestyle intervention with the 500 without any treatment 500 persons treated with both MET and Iifestyle intervention with the 500 withaut any treatment.
In ancitlary companion studies we will analyse the epigenetio pattern al baseline, and its change over one year of treatments, in participants who developed, or did not develop, ArCD, as well as in participants who succeeded, or did not succeed, to make MetS regress (with or without MET or lifestylo changes). 1510512013 Power computations have been based a) on expected mortality and ArCD incidente in the study area in -tnend women aged 55 or more (assuming the same age distribution as the generai population), 1) on the assumption that MetS is associated with 1,5 higher incidence of ArCD (3 times more for diabetes); o) on the hypothesis that the pianned interventions may reduce ArCD incidente by 25 or 33%; d) on a 35year average follow-up (within the 5-year study period); and, e) alpha error = 0.05. 1-lypothesising to recruit 1000 persona with MS per year in The first Mo years, excluding the person-years accumulated in the recruitment year, plus 200 persona lo compensate the power ]osa due to expected attrition (for mortality and decisions lo stop participation), we would expect about 174 deaths, 175 cancers, and 119 major cardiovascular events (myocardial infarotion and stroke), and 219 casca of diabetes, summing up to over 500 incident casca of major ArCD.
We will have, for 33% (and, respectivs!y. 2591 ) reducUon in tbc cumulative incidence of major .ArCD (cardiovascular + diabetes 4cancer): -99% (92%) power te compare 1,000 persona treated with mefformin with 1,000 receiving placebo (or 1,000 persona with or without attive lifestvle intervention) -97% (84%) power to compare 500 persona treated with both metiormin and Iifestyle intervention with respect to the other 1,500. 89% (65%) power to compare 500 persons with both interventions with 500 with none Comparing 1,000 treated and 1,000 non-treated persona, we will have 69% power lo detect a signifloant 33% differente in total mortality, or in the cumulative incidence of cancer, and 98% power lo detect a 33% difference in the cumulative incidente of diabetes (85% for 25% reduotion).

c. Resources
The Fondazione IRCCS Istituto Nazionale dei Tumori is fiilly equipped with infrastructures for gymnasium, dedicated kitchen and cafeteria, teaching rooms, blood soliection and sample storage, laboratories for biochemical assays. As the hlost Institution has noi sufticient qualitled personnel br kitohen activities, theses activities as well as The purchase and stocking of food will be assigned io a subcontractor, an agency with knowedge of Mediterranean and Tvlacrobiotic diet and whose tasks w111 be to buy and prepara meals according to the instructions of the research team, and to teach kitchen technicalities. The meeting will include; • clinica! visit • expianation on how te take the pilis; • in the Ilrst 15 days the participant must take half a p111 (425mg) of metformin per day, during a main meli! (breakfast, Iunch or dinner) • in the subsequent 15 days the participant musttake a fui[ pili (850m9) of metformin per day, during a main meal (breakfast, lunch or dinner) • discussion of the possible side effects and invitation to contact the study physician, available 2411124, in case of syniptoms or questions • if The participant miss taking one or more pilis, he/she must noi increase the dose in the foliowing days, and must not throw away the unused pills, which must remain in the box and rendered io the study centre ai the next meeting • arrangernent of the next meeting (after 30 days) • each participant is given a leaflet with instructions a fl MeMeMe 322752 15/0512013 Fourth meeting P. Evaluation of compliance ami side effects to complete the eligibility cl%tkst.AJ2 -30 days after the start of metforniin treatment, the physician will check the participant's compliance ttirough a) the counting of residual pilis b) specifio questions un side effects and on wiliingness te continue Participants having demonstrated an adequate compliance and no or trivial side effects will be randomized RANDOMIZATION Fifth meeting: Participants assume a placebo or mettormin (650 mq) p1111 per day far 30 days.
The physìcian will: • Provide the pilis for the next 30 days • Remember notthrowing away the unused pilis • Arrange the next meeting ([hai wUl be aiso remembered trough an SMS) • Arrange the dates of kitchen courses for participants randomized in the active Iifestyle intervention group Sixth meeting: Participants start taking metformin or placebo pilis twice a day for mie month The physician will • Collect information on side etlects.
• Gount the non used pills io verity compliance.
• Provide [ho pilis for the next 6 months. Participants shall assume two pilis per day (for a total of 1700mg of metformin in the intervention ami); il is recomrnended io take the pili during the main mears (e.g. one ai breakfast and one ai dinner, or one ai Iunch ami one ai dinner) • Remember netto increase the dose ami noi throw away the unused pilis • Stop taking the pilis for a few days in case of surgical interventions, anaesthesia, or radiological examinaions requiring oontrast means • Arrange the next meeting • Arrange file dates of common iunches or dinners and for physicaI exercise meetings for participants randomized in the active Iifestyle intervention group lth enti Bth meeting: Participants, in absence of major side effects, receives the pilis for the next 6 months. li the fui] dose is noi tolerated participants ara invited io go back io a singie pili per day. 91h meeting: After 12: months of taking metformin or placebo ai fuli dose, participants ara invited far a new clinical viali, eligibility check, anthropometric measurements ami blood ami urine sample. In case of contr.indication io continue metformin treatment (eg. for intervening diagnosis of renal insufficiency or major cardiovascular, metabolio or respiratory diseases) participants will be reconimended te interrupt the drugs and to continue only the dietary intervention. The participants generai practitioner will be infommed.If there ere no contra-indications the participants will be given the pilis for the next 12 months. Ai every meeting participants aro invited io fili in a food frequency ami physical activity questionnaire; this will allow the study dietician io reinforce Iifestyle recommendations for those randomized in the active Iifestyie intervention group.Participants randomized in the intervention group not showing signiticant improvernents will be offered special teaching sessions.
Furtheryearly meeting: To check eligibility, body weight ami waist circumference, ami io provide the pilis for the next 12 ma n th s Every meeting will be preceded by a telephone rernind (SMS) Ai each meeting ami in a random day every two months ali participants will be invited (through SMS or teiephone) io fili in a 24h food frequency and physicaI activity diary Far participants randomized into the dietary intervention group the meetings wilil usuafly arranged in the sane days ofthe kitchen courses and common meais. Participants who for whatever reason decide io interrupt The study wili be invited for a final olinical visit and anthropometric measurement a MeMeMe 322752 1510512013 e. Funding Amount of funding necessary The esimates are Iargely based on v4iat we spent for our ongoing Ufe-style intervention study of 2,000 breast cancer survivors.

Drug
On average participants will be treated far 4 years with either Metformin or placebo. Our pharmacist has estiniated that we will need 400,000 Euro to prepara Metformin and placebo far the whole study. Metforniin is very cheap (Iess than 5 Euro per month oftreatment) but companies aro noi interested to sponsor a study.
Food and kitchen activitios On average participants randomized in the intervention group will receive 4 fiulkday kitchen courses and villi be invited monthly far Iunch or dinner with short kitchen courses for 4 years (IO months per year), for a totai of 40,000 meals. In previous trials we spent 7 Euro per nieal. Taking into account infiation but also some scale economy the totai expenditure far food and kitchen activities wili ho 8 x 40,000 = 320,000 Euro (125,000 aboutforfood purchase).
Laboratory activities Laboratory costs aro mainly far basic clinicai ohemistry and insulin, growth factors, and inflamrnatory cytokines. Far epigenetictests we have appliedto othersources.